生物医药

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, characterized by genetic heterogeneity and the accumulation of mutations in key oncogenes and tumor suppressor genes. CRISPR-Cas9 technology has greatly advanced genetic research by enabling precise genome editing. This review focuses on the innovative applications of CRISPR-Cas9 in CRC research, particularly its role in identifying novel therapeutic targets, elucidating mechanisms of drug resistance, and uncovering metabolic and stem cell pathway alterations in tumorigenesis. We highlight the diverse CRISPR systems, including Cas9, Cas12, Cas13, and advanced variants such as CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), base editing, and prime editing, which have expanded gene knockout studies and enhanced our understanding of CRC. Despite these breakthroughs, challenges such as off-target effects and delivery limitations remain. Ongoing efforts to refine CRISPR technology aim to enhance its precision and clinical applicability, ultimately paving the way for more effective and personalized treatment strategies for CRC. In this review, we explore these advances and focus on the latest developments in CRISPR-based approaches for CRC treatment.

Antimicrobial resistance (AMR) has escalated into a global health crisis, with resistant pathogens causing over 1.2 million direct deaths annually and threatening to render modern medicine unsustainable. This review provides a comprehensive and updated synthesis of CRISPR-Cas-based antimicrobial strategies with a unique focus on: (i) critical comparison with conventional antibiotics and emerging alternatives; (ii) quantitative evaluation of delivery platforms; (iii) novel strategies including AI-optimized guide design and the ATTACK-CreTA system; (iv) comprehensive analysis of ecological risks; and (v) technology readiness level assessments for clinical translation. The CRISPR-Cas system, originally discovered as a bacterial adaptive immune mechanism, has been repurposed as a programmable precision tool to combat AMR by selectively targeting and eliminating resistance genes. We systematically evaluate the mechanistic diversity of Cas effectors, from DNA-cleaving Cas9 and Cas3 to RNA-targeting Cas13, and their application in reversing resistance phenotypes in WHO priority pathogens. We critically assess emerging delivery platforms, including engineered bacteriophages, conjugative plasmids, nanoparticles, and outer membrane vesicles, quantitatively comparing their delivery efficiency, payload capacity, and biosafety profiles. Novel strategies such as CRISPR interference (CRISPRi) for gene silencing without genomic cleavage, the ATTACK-CreTA system for enhanced bactericidal activity, and AI-driven optimization of guide RNA design are examined with appropriate caveats. We comprehensively address clinical translation challenges including immunogenicity, pharmacokinetics/pharmacodynamics, manufacturing scalability, regulatory pathways, and bacterial resistance mechanisms including anti-CRISPR proteins. No CRISPR-based antimicrobial has yet received regulatory approval, and we critically evaluate the gap between proof-of-concept and clinical utility. A detailed roadmap for clinical development is proposed. By integrating recent advances in Cas protein engineering, delivery technologies, and diagnostic applications, this review positions CRISPR-Cas systems as next-generation precision therapeutics capable of both treating resistant infections and curtailing the spread of AMR across clinical and environmental settings.

Decapod iridovirus 1 (DIV1) is a highly lethal pathogen that infects decapod crustaceans including Litopenaeus vannamei, causing mass mortality in cultured shrimp and severe economic losses worldwide. The ATPase gene is a highly conserved region within the DIV1 genome, plays a critical role in viral replication and represents an ideal target for molecular diagnostic development. In this study, we established a rapid, sensitive and field-adaptable detection platform for DIV1 by integrating recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system. RPA enables efficient isothermal amplification of target nucleic acids, achieving exponential enrichment of the target nucleic acids and exerting the function of signal amplification. While the CRISPR/Cas12a system upon crRNA-guided specific recognition of the amplicon, triggers robust trans-cleavage activity against reporter probes for signal generation and readout. After systematic optimization, the RPA reaction was performed at 38°C for 10 min and the CRISPR-Cas12a reaction was conducted at 37°C for 20 min. The integrated two-step workflow completed detection within 40 min, with a limit of detection of 2.3 × 101 copies/μL. Specificity evaluation confirmed that the RPA-CRISPR/Cas12a assay exclusively recognised DIV1 without cross-reaction with other major shrimp pathogens. Further validation using clinical shrimp samples demonstrated stable and reliable performance, supporting its practical utility in aquaculture settings. In conclusion, the established CRISPR/Cas12a-based detection platform provides a robust technical tool for early warning and on-site rapid screening of DIV1, facilitating timely disease control and risk management in shrimp farming.

Highly sensitive nucleic acid detection is essential for analytical applications. Conventional methods often require complex pre-amplification procedures, limiting their practical utility in screening. Developing nucleic acid detection strategies with high sensitivity and selectivity, without target gene pre-amplification, remains a significant challenge. This study integrates CRISPR/Cas12a recognition, catalytic hairpin assembly (CHA) amplification, and surface-enhanced Raman spectroscopy (SERS). CRISPR/Cas12a recognizes target nucleic acids and cleaves single-stranded DNA (ssDNA), thereby blocking the toehold-mediated strand displacement reaction (TSDR) and triggering CHA. Hairpin probe HP1 with C-Ag+-C structures bridges CHA, releasing Ag+ through cyclic amplification. Ag+ induces charge transfer and aggregation of AgNPs@4-ABT, generating strong SERS signals. The platform achieved femtomolar sensitivity and high selectivity in detecting pCaMV35S, with 96.4% accuracy in maize seeds and 100% in maize leaves. This strategy eliminates the need for pre-amplification of target genes by combining CRISPR's targeting feature, CHA, and ultrasensitive SERS detection. It demonstrates excellent performance in genetically modified organism screening, seed quality testing, and leaf sample analysis, providing a promising tool for food safety and agricultural regulation.

Avoidable prostate biopsies remain a persistent weakness of prostate specific antigen (PSA)- and imaging-led prostate cancer (PCa) diagnosis. The key need is a non-invasive test that improves pre-biopsy risk stratification while remaining potentially translatable to clinical deployment. We developed and validated an end-to-end liquid-biopsy pipeline linking serum miRNA markers, routine clinical variables, machine learning, and CRISPR/Cas13a-based detection. Candidate miRNAs were prioritized from GSE112264 by differential expression, Logistic Regression, and Least Absolute Shrinkage and Selection Operator analyses, cross-referenced with PCa tissue expression, and measured by qPCR in 712 biopsy-scheduled participants from Sun Yat-sen Memorial Hospital (SYSMH), Houjie Hospital of Dongguan (HHD), and Ganzhou People's Hospital (GPH). A three-miRNA PCa risk score (PCaRS) was trained in SYSMH and tested in internal, external, and prospective cohorts. PCaRS and independent clinical predictors were integrated using six machine-learning algorithms; the optimal model was selected by receiver operator characteristic and DeLong analyses. Finally, serum miRNAs in the prospective SYSMH-Pro cohort were quantified with polydisperse droplet digital CRISPR/Cas13a (PddCas13a) to assess whether a CRISPR/Cas13a readout could support a practical miRNA-based diagnostic workflow. Three serum miRNAs (miR-17-3p, miR-504-3p, and miR-6877-5p) were identified as diagnostic markers. PCaRS achieved stable discrimination across the SYSMH Train, SYSMH Test, HHD, and GPH cohorts [AUCs: 0.836 (0.790 - 0.881), 0.832 (0.773 - 0.907), 0.826 (0.721 - 0.932), and 0.820 (0.702 - 0.938), respectively]. PCaRS, f/tPSA, PSA Density, and Prostate Imaging Reporting and Data System score were independent predictors of PCa. Among six machine-learning models, the Support Vector Machine based composite model (PCaSVM) achieved the best performance, with AUCs of 0.939 (0.912 - 0.966), 0.899 (0.849 - 0.948), 0.886 (0.806 - 0.967), and 0.905 (0.834 - 0.976) in the four retrospective cohorts and 0.873 (0.772-0.975) in the prospective cohort. In the prospective cohort, a PddCas13a-derived score (PCaCas13aS) achieved an AUC of 0.831 (0.783 - 0.872), with no significant difference from the qPCR-based PCaRS. The PCaSVM achieved satisfactory diagnostic performance, suggesting potential utility for non-invasive diagnosis of PCa. The PddCas13a-based quantitative detection of serum miRNAs presents a feasible approach for diagnosing PCa. Larger prospective multicenter studies are warranted to confirm biopsy-sparing clinical utility.

Recurrent crop disease outbreaks linked to global warming pose challenges to sustainable food production. Conventional plant breeding techniques may become less effective at addressing these threats, as improving disease resistance often causes yield reduction. Amid these challenges, CRISPR/Cas-based gene editing offers targeted and tractable solutions. This review synthesizes recent approaches to uncoupling immunity from productivity in cereals. We show that susceptibility (S) gene disruption can provide resistance without activating costly defense mechanisms. We also discuss the generation of new alleles through targeted modifications that mitigate autoimmunity-associated fitness costs. Here, we propose CRISPR-mediated de-moonlighting, an approach for decoupling multifunctional protein activities. Multiplex editing of minor resistance loci, especially in polyploids such as wheat, offers long-lasting, broad-spectrum protection. These strategies converge on the manipulation of canonical moonlighting proteins, multifunctional signaling hubs, and pleiotropic regulators, which serve as regulatory nodes that link development and immunity. CRISPR-mediated precision modification of these regulators can fine-tune the growth-defense balance. Combining these approaches with systems biology, AI-driven design and advanced breeding pipelines can help develop high-yielding, disease-resistant cereals for sustainable agriculture.

Despite advances in therapy, arterial, venous, and pulmonary vascular diseases remain leading causes of morbidity and mortality. Persistent endothelial dysfunction, inflammation, oxidative stress, and maladaptive vascular remodeling continue to drive disease progression and residual risk. CRISPR/Cas9 technology offers a unique opportunity to modify the molecular pathways underlying vascular pathophysiology directly. The PRISMA 2020 guidelines guided the systematic review. The databases PubMed/MEDLINE, Embase, Web of Science, Cochrane Library, ClinicalTrials.gov, and Google Scholar were searched from their inception until September 2025 for experimental and/or clinical studies evaluating the application of CRISPR/Cas9 on vascular disease. Included were in vitro studies, animal model studies, and early-phase human studies aimed at targeting the endothelial cell regulatory pathways, inflammatory pathways, metabolic remodeling processes, and hereditary causes of vasculopathy. Seventeen studies met the inclusion criteria. CRISPR technologies targeting PCSK9, NOS3, HIF1A, NLRP3, METTL4, BMPR2, and ACTA2 were identified to enhance repair mechanisms in endothelial cells, regulate inflammation, modulate lipid metabolism, and remodel the vascular system. The human studies demonstrated sustained gene silencing effects following a single dose of CRISPR-induced in vivo editing. The use of CRISPR technology to edit cell genomes offers potential to alter disease progression in vascular medicine, with a growing body of translational evidence supporting the feasibility and durability of the approach.

An in vitro CRISPR/Cas9-RNP system efficiently cleaves CcYUC1 in Coffea canephora, establishing a foundation for DNA-free genome editing in coffee. Somatic embryogenesis depends on auxin biosynthesis and signaling; however, functional validation of candidate genes remains limited. In this study, we identified CcYUC1, a putative flavin monooxygenase gene associated with indole-3-acetic acid biosynthesis, during SE induction in Coffea canephora. CcYUC1 transcripts accumulated during the early stages of SE, suggesting a role in embryogenic induction. To establish a genome editing platform in coffee, we designed a CRISPR/Cas9 ribonucleoprotein (RNP) system targeting exon 4 of CcYUC1. In vitro cleavage assays confirmed specific and efficient digestion of the target, achieving over 80% cleavage under optimized Cas9/sgRNA conditions. These findings establish a proof-of-concept CRISPR/Cas9-RNP platform for DNA-free genome editing in coffee and provide a basis for future functional studies of auxin biosynthesis during somatic embryogenesis.

Type I CRISPR-Cas systems constitute the most prevalent prokaryotic adaptive immune pathways and are classified into seven subtypes (I-A to I-G). These antiviral systems typically exploit a Cascade complex for RNA-guided DNA recognition and a Cas3 helicase-nuclease effector for DNA degradation, yet their diverse activation mechanisms remain not fully understood. In this study, we isolate the I-A Cascade from Saccharolobus islandicus, revealing a minimal form of Cascade lacking both Cas3 and the CRISPR-RNA maturase Cas6. Cas3 is recruited to the R-loop structure formed after Cascade binding to target DNA, which activates the effector for both cis- and trans-DNA cleavage. Strikingly, ATP not only enables the processive target degradation by Cas3 but also suppresses the trans-cleavage of the same enzyme. Together, the Sa. islandicus I-A system operates via target-dependent Cas3 recruitment-a mechanism distinct from other characterized I-A systems, thus underscoring the mechanistic diversity within type I CRISPR-Cas immunity.

Lipopolysaccharide (LPS) initiates hyperinflammatory cascade via the LBP-CD14-MD2-TLR4 complex which can precipitate fatal cytokine storms and septic shock. We aim to develop molecular strategies that selectively dampen TLR4-driven inflammation without eliminating host defense. Here, we demonstrate that CRISPR-Cas9-mediated dual-site editing of the TLR4 gene, by introducing specific disruptions in both the extracellular domain (ECD) and the intracellular Toll/IL-1 receptor (TIR) domain, can generate a partially uncoupled signaling phenotype that selectively attenuates acute inflammation while preserving baseline stress-adaptive mechanisms. By combining whole-genome and amplicon-based next-generation sequencing, RNA-Seq, molecular dynamics simulations, transmission electron microscopy (TEM), GSEA, and functional reporter assays, we show that targeted mutations near the LPS-binding interface within the LRR modules of the ECD (M209I, V254I, E593D) and within the CD loop of the TIR domain (R761H) reduce ligand binding-pocket volume by ~17% (688.19Å3 vs. 825.41Å3) within TLR4 loci, leading to partial signal propagation and altered LPS trafficking to lysosomal compartments through autophagosome sequestration. Immunofluorescence profiling revealed broad attenuation of TLR-signaling networks, MyD88/TRAF6 recruitment, and downstream NFκB-MAPK-PI3K-AKT-JAK-STAT-mTOR cascades alongside decreased CD14 expression, suppressed ROS generation, and diminished caspase-3 activity. While complementary NFκB and LC3-HiBiT reporter assays confirmed interrupted LPS-induced inflammatory transcription and conventional autophagic flux activation, TFEB reporters revealed that edited macrophages remain highly sensitive and responsive to direct metabolic mTOR-dependent metabolic inhibition via Rapamycin. Collectively, our findings establish TLR4 as a central molecular switch and suggest that CRISPR-Cas9-mediated dual-site editing reprograms macrophages into repair-oriented, adaptive phenotype with implications for therapeutic strategies targeting inflammation, sepsis, and autophagy-driven tissue protection.

Metallothionein 4 (MT4) is a low-molecular-weight, cysteine-rich metal-binding protein belonging to the metallothionein family. It exhibits unique skin developmental and differentiation inhibitory activity when functionally impaired and regulates skin cell growth and disease through multiple mechanisms. However, its exact role in cell fate determination remains unknown. Here, we utilized the CRISPR/Cas9 system to generate a homozygous Mt4 knockout (Mt4-/-) mouse embryonic stem cell (mESC) line. This cell line maintains normal morphology, pluripotency, and the ability to differentiate into all three germ layers. It provides a valuable resource for investigating the mechanisms underlying skin diseases caused by MT4 gene mutations.

Traumatic brain injury (TBI) triggers complex secondary pathologies that lack effective treatments. While mesenchymal stem cell (MSC) transplantation is promising, it is severely limited by poor cell retention and survival. To address these challenges, we engineered a combinatorial platform comprising an injectable, self-healing supramolecular gelatin hydrogel (iGel) loaded with CRISPR-SAM-engineered "Super MSCs" (SPMSCs). These cells were programmed to endogenously multiplex the activation of neuroprotective factors IL-10 and FGF21. Our results demonstrate that the biomimetic iGel niche enhances SPMSC viability and sustained factor secretion compared to 2D cultures. In a murine TBI model, iGel-encapsulated SPMSCs exerted potent immunomodulatory effects, suppressing microglial inflammation and neuronal apoptosis while restoring blood-brain barrier integrity. Furthermore, the treatment promoted angiogenesis and endogenous neurogenesis. Consequently, treated mice exhibited reduced cerebral edema and lesion volume, alongside significant improvements in sensorimotor function and spatial memory. This study establishes a versatile, gene-editing-empowered biomaterial platform that overcomes critical bottlenecks in cell therapy for central nervous system injuries.

The precise enhancement of nutritional quality in silage maize is a core strategy for increasing livestock production efficiency. Through evolutionary analysis of multiple plant species, we identified two functionally synergistic upstream open reading frames (uORF1 and uORF2) within the 5' untranslated region of the gene encoding GDP-L-galactose phosphorylase (GGP). By leveraging a natural translation-enhancing haplotype of uORF1 (Hap2) and performing CRISPR/Cas9-mediated targeted mutagenesis of the highly conserved uORF2, we successfully engineered an elite dual-uORFs variant. This variant significantly increased vitamin C (Vc) content and concurrently improved key silage quality traits, including crude protein and phosphorus levels, without inducing growth penalties. Transcriptomic profiling further elucidated the molecular mechanisms by which the dual-uORFs variation coordinately regulates Vc biosynthesis and the improvement of silage quality. Our findings deepen the understanding of the conventional paradigm of single-uORF regulation and provide a novel strategy and superior germplasm resources for the precision breeding of high-Vc, high-quality silage maize.

Urinary tract infections are one of the most common infectious diseases, with Escherichia coli as the predominant pathogen. Traditional diagnostic methods fail to meet clinical demands for rapid and specific detection. Here, we developed an efficient urine E. coli detection strategy via a reporter phage cocktail. Four reporter phages (T2::Nluc, T4::Nluc, T5::Nluc, T6::Nluc) were constructed by the CRISPR/Cas9 system combined with homologous recombination. One-step growth curves, optimal multiplicity of infection, and lytic efficiency showed that the Nluc gene block insertion exerted heterogeneous effects on phages. Luminescence assays demonstrated that all five reporter phages (including previously preserved T7::Nluc) and the cocktail offered favorable limits of detection (≥103 CFU/mL), high specificity, and no urine matrix interference. However, single phages exhibited limited coverage among 177 clinical E. coli isolates. But the reporter phage cocktail remedies this limitation. In large-scale clinical validation, the cocktail achieved sensitivity 73.15% (63.76%-81.22%), specificity 100.00% (99.53%-100.00%), positive predictive value (PPV) 100.00% (95.44%-100.00%), and negative predictive value (NPV) 96.42% (95.18%-97.36%) (all 95% confidence interval [CI]), and excellent concordance with the gold-standard method (Kappa = 0.83, 95% CI: 0.77-0.89), greatly outperforming single reporter phages (~40.00% sensitivity). This method requires no sample pretreatment, is simple to operate, and completes detection within 4 h, significantly improving diagnostic efficiency. Accordingly, it provides a novel platform for pathogen detection and supports the clinical translation of reporter phage diagnostics.IMPORTANCEUrinary tract infections impose substantial economic and public health burdens. In this study, we successfully constructed Escherichia coli-specific reporter phages T2::Nluc, T4::Nluc, T5::Nluc, and T6::Nluc. Combined with the previously preserved T7::Nluc, these phages formed a reporter phage cocktail. Co-cultivation of this cocktail with clinical samples enabled rapid and specific detection of E. coli in clinical urine, with a significantly shortened detection time (4 h) and good concordance with the gold-standard detection method (Kappa = 0.83), effectively improving detection efficiency and accuracy. This novel pathogen detection platform, integrating specific recognition and signal amplification, not only provides a new technical approach for the rapid and accurate diagnosis of clinical urinary tract infections but also effectively promotes the coordinated improvement of infectious disease diagnosis and treatment in terms of timeliness-precision-cost.

Weeds significantly threaten rice production. While herbicides are the most efficient weed-control method, prolonged use accelerates resistant weed emergence. 4-Hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides offer potential for managing resistant weeds in rice, but the availability of resistant rice mutants is limited. This study aimed to optimize the method of DNA shuffling to conduct directed evolution of the rice HPPD gene, discover novel resistance mutations, and generate herbicide-resistant rice germplasm. We developed an efficient DNA shuffling method utilizing flanking sequences and segmented amplification to construct random mutation libraries of rice HPPD and maize (Zea mays L.) HPPD genes. Mutant libraries were screened via chromogenic reaction in Escherichia coli. CRISPR-mediated adenine base editing was adopted to generate rice mutants, and hydroponic assays were conducted to evaluate herbicide resistance. The crystal structure of rice HPPD was determined by X-ray crystallography, while mutant structures and ligand docking were simulated with AlphaFold3. Flanking sequences enhanced single-stranded DNA amplification efficiency, and segmented amplification reduced nonsense mutations. A resistant mutant, OsHPPD-mHelix, was obtained. Enzyme kinetics analysis demonstrated that its Ki for mesotrione was increased approximately 2.5-fold compared to that of wild-type OsHPPD, while its enzyme activity was not significantly affected. Using the mutation sites contained in OsHPPD-mHelix as hotspots, genome-edited rice was further generated. It was found that the mesotrione resistance of rice carrying the E423G mutation was approximately 2.2-fold higher than that of wild-type rice. Structural and molecular dynamics analyses suggested the E423G mutation promotes a closed terminal α-helix conformation, with free energy landscapes indicating reduced mesotrione binding stability, potentially explaining the resistance mechanism. This study provides an efficient DNA shuffling technology and novel herbicide-resistant rice germplasm, enriching the toolbox for rice gene-directed evolution and resistant variety cultivation.

Small extracellular vesicles (sEVs) have emerged as promising liquid biopsy biomarker for gastric cancer (GC) diagnosis. However, profiling sEVs surface proteins in complex clinical matrices remains challenging by limited sensitivity and high background interference. In this study, we developed an electrochemical platform based on the photoactivated liquid-liquid phase separation cascaded Cas12a amplification (PL-cCas12a) for ultrasensitive and specific detection of sEVs surface proteins. The rationally designed system enables precise spatiotemporal control over the reaction. Photoactivation ensures precise reaction initiation. The spatially confined microenvironment accelerate reaction efficiency by local substrates enrichment and suppresses non-specific trans-cleavage leakage via steric hindrance. The PL-cCas12a platform achieved the limit of detection (LOD) as low as 3.95 × 103 particles/μL with negligible background leakage. By integrating the aptamers targeting GC-associated sEV surface proteins, this platform accurately discriminated GC patients from healthy donors (AUC = 0.912). Therefore, this platform represents a promising tool for non-invasive early cancer diagnosis.

Diffuse large B-cell lymphoma (DLBCL) can be subclassified by phenotype into germinal center B-cell-like and activated B-cell-like (ABC) subtypes and by recurrent potentially oncogenic mutations into 5 to 7 genetic clusters. In ABC-DLBCL, potentially oncogenic mutations frequently occur in genes involved in B-cell receptor (BCR) signaling and NF-κB activation. Autonomous BCR signaling acts as an alternative immunologic driver predominantly in ABC-type DLBCL that cannot be captured by either subclassification system. The relative functional contribution and interdependence of these mechanistically diverse oncogenic drivers have not been completely defined. To directly compare the effects of autonomously signaling BCR and signalosome-activating CARD11 mutations on NF-κB activation and survival of ABC-DLBCL, we reciprocally exchanged these driver mechanisms in the MYD88L265P-mutated ABC-DLBCL cell lines TMD8 and OCI-Ly3. Only CARD11L251P (not CARD11K215N, CARD11D230N, and CARD11R337Q) compensated TMD8 cells for the loss of autonomous BCR signaling, as indicated by survival of BCR knockout and conversion to complete resistance to acalabrutinib. Transduction of the TMD8 BCR rescued OCI-Ly3 cells from replacing the CARD11L215P variant with CARD11WT. The autonomous TMD8 BCR signal provided a slight growth advantage over CARD11L251P-driven cells in both reciprocal systems. Unsupervised clustering of genetically engineered TMD8 and OCI-Ly3 clones demonstrated tight clustering with their parental cells and only minor alterations of cellular pathways. Only the strongest signalosome-activating mutation has functional near-equivalency to an autonomously signaling BCR for NF-κB activation and growth and survival in ABC-DLBCL. Quantifying the effects of co-occurring potential NF-κB-activating mechanisms is essential to predict Bruton tyrosine kinase (BTK) inhibition sensitivity in individual ABC-DLBCL cases.

CRISPR/Cas12a has emerged as a powerful tool for nucleic acid detection; however, its clinical utility is severely hampered by intrinsic limitations in single-nucleotide variant (SNV) discrimination, reliance on pre-processed single-stranded DNA (ssDNA) templates, and cumbersome multi-step workflows. Here, we report a novel molecular design principle by engineering crRNAs with site-specific dual mismatches (positions 12 and 14 relative to the PAM), which we systematically demonstrate to drastically enhance the SNV discrimination capability of Cas12a. Leveraging this breakthrough, we developed FOCUS (Fast One-step CRISPR-based Universalizable SNV detection system), an all-in-one CRISPR sensing platform that enables isothermal detection of SNVs from double-stranded DNA (dsDNA) by integrating amplification and detection in a single reaction system. FOCUS achieved attomolar-level sensitivity (13.15 aM) and ultrafast readout (< 20 min) for distinguishing the highly homologous survival motor neuron 1 (SMN1) and SMN2 genes-the gold standard challenge for SNV genotyping in spinal muscular atrophy (SMA) diagnostics. To validate its clinical translatability, FOCUS was successfully adapted to a low-cost, equipment-free assay using lateral flow strips and UV visualization, facilitating point-of-care testing (POCT). In a comprehensive validation across 175 clinical samples, FOCUS exhibited 100% diagnostic concordance with gold-standard methods for SMA (21 samples), high-risk HPV 16/18 (27 samples), Staphylococcus aureus (20 samples), and SARS-CoV-2 (107 samples). Collectively, our study establishes a generalizable engineering strategy for Cas12a crRNAs and presents FOCUS as a robust, versatile, and field-deployable solution for precision SNV genotyping, underscoring the translational medicine value of FOCUS in molecular diagnostics.

Bovine viral diarrhea virus type 1 (BVDV-1) is an RNA virus known for lifelong persistent infection in affected animals and substantial economic impact in cattle. While current approaches of infection control rely on biosecurity and immunization, recent advances in genome editing provide opportunities for improving genetic resistance of livestock to viral diseases. Any improvements in our understanding of the role of host genes in viral replication could lead to new gene-editing applications, supporting future efforts to control BVDV-1 worldwide. Recent evidence demonstrates that Synaptogyrin-2 (SYNGR2) gene plays a role in the replication of DNA and RNA viruses across different hosts, from humans to pigs. Given the functions of SYNGR2 in pathogen entry and viral replication, this study aimed to assess the role of this gene in BVDV-1 infection. The BVDV-1 strain used in the study was propagated in Madin-Darby bovine kidney (MDBK) cells and sequenced using Oxford Nanopore Technology. The sequencing reads were subjected to de novo genome assembly, exhibiting substantial similarity with Singer Arg BVDV-1 strain (97.75%). A SYNGR2 knock-out (KO) MDBK clone was generated via CRISPR-Cas9 gene editing. The KO clone included a 163&#x2009;bp deletion in the mRNA of the SYNGR2 gene, predicted to result in a shift in the reading frame starting after residue 118, affecting the second intraluminal loop and the carboxyl terminal domain. There was no difference in viral replication between wild type and SYNGR2 KO MDBK clone, measured in cells (P > 0.08) and supernatant (P > 0.21), suggesting that SYNGR2 is less likely critical for the replication of BVDV-1. Our findings could be a result of key differences from previous findings that involved SYNGR2 in the replication of DNA and RNA viruses, emphasizing the need for exploring in more detail the role of host genes in BVDV-1 replication and pathogenesis. Bovine viral diarrhea virus type 1 (BVDV-1) is a widespread viral infection responsible for large economic losses in cattle herds. In this study, a knock-out (KO) gene edited cell line targeting the Synaptogyrin-2 (SYNGR2) gene was generated via CRISPR-Cas9 to test the role of this gene in BVDV-1 replication. Following in vitro BVDV-1 infection of wild type and SYNGR2 KO clones, no differences in viral titer were observed between clones, measured in cells (P > 0.08) and in the supernatant (P > 0.21). This finding is indicating that SYNGR2 is potentially not essential for the replication of BVDV-1.

Pancreatic ductal adenocarcinoma (PDAC) exhibits profound metabolic plasticity that underlies its aggressive growth and therapeutic resistance. However, genetic determinants that modulate PDAC sensitivity to glycolytic inhibition remain incompletely defined. Here, we performed a pooled genome-wide CRISPR-Cas9 dropout screen in PDAC cells under 2-deoxy-D-glucose (2-DG) selection and identified FGD5 as a key regulator of glycolytic dependency. Loss of FGD5 sensitized PDAC cells to 2-DG, reduced clonogenic growth and stem-like properties, and was associated with a shift from glycolysis toward oxidative phosphorylation. Clinically, FGD5 expression was elevated in PDAC tissues and correlated with unfavorable patient outcomes. Mechanistically, FGD5 interacted with and stabilized PGK1 by limiting STUB1-mediated ubiquitination and proteasomal degradation. Pharmacological perturbation with CB-5083, a compound prioritized through structure-guided screening, modulated FGD5-PGK1-associated proteostasis readouts, increased PGK1 ubiquitination, reduced PGK1 abundance, and cooperated with 2-DG to suppress PDAC growth in vitro and in vivo. Moreover, lactate-associated H3K18 histone lactylation was linked to increased FGD5 transcription, consistent with a feed-forward regulatory connection between glycolysis, epigenetic modification, and metabolic adaptation. Collectively, our findings position FGD5 as a metabolic vulnerability in PDAC and provide a mechanistic framework supporting combinatorial strategies that couple glycolysis inhibition with perturbation of FGD5-PGK1-associated proteostasis. A genome-wide CRISPR-Cas9 screen identified FGD5 as a metabolic vulnerability that increases PDAC sensitivity to the glycolysis inhibitor 2-deoxy-D-glucose (2-DG). In PDAC cells, FGD5 interacts with PGK1 and supports PGK1 stability by limiting STUB1-dependent ubiquitination and proteasomal degradation, thereby sustaining aerobic glycolysis and lactate production. Glycolysis-derived lactate is associated with increased H3K18 histone lactylation (H3K18la) at the FGD5 promoter, accompanied by elevated FGD5 transcription, consistent with a metabolic-epigenetic regulatory link. Pharmacological perturbation with CB-5083 attenuates FGD5-PGK1-associated proteostasis readouts, whereas 2-DG inhibits glycolytic flux and lactate-associated H3K18la. Together, CB-5083 and 2-DG cooperate to suppress PDAC growth by coupling perturbation of the FGD5-PGK1-associated proteostasis axis with glycolytic inhibition.